Serum IGFBP-1 as a promising diagnostic and prognostic biomarker for colorectal cancer

Our previous study showed that levels of circulating insulin-like growth factor binding protein-1 (IGFBP-1) has potential diagnostic value for early-stage upper gastrointestinal cancers. This study aimed to assess whether serum IGFBP-1 is a potential diagnostic and prognostic biomarker for CRC patients. IGFBP-1 mRNA expression profile data of peripheral blood in colorectal cancer (CRC) patients were downloaded and analyzed from Gene Expression Omnibus database. We detected serum IGFBP-1 in 138 CRC patients and 190 normal controls using enzyme-linked immunosorbent assay. Blood IGFBP-1 mRNA levels were higher in CRC patients than those in normal controls (P = 0.027). In addition, serum IGFBP-1 protein levels in the CRC group were significantly higher than those in normal control group (P < 0.0001). Serum IGFBP-1 demonstrated better diagnostic accuracy for all CRC and early-stage CRC, respectively, when compared with carcinoembryonic antigen (CEA), carbohydrate antigen19-9 (CA 19-9) or the combination of CEA and CA19-9. Furthermore, Cox multivariate analysis revealed that serum IGFBP-1 was an independent prognostic factor for OS (HR = 2.043, P = 0.045). Our study demonstrated that serum IGFBP-1 might be a potential biomarker for the diagnosis and prognosis of CRC. In addition, the nomogram might be helpful to predict the prognosis of CRC.


ELISA for IGFBP-1
The expression levels of serum IGFBP-1 were measured by a commercial ELISA kit (CUSABIO, Wuhan, China), and the procedure was carried out according to the manufacturer's recommendations in the ELISA kit.Briefly, 100 μl of serum sample at 20 fold-dilution and standard were added into antibody-coated 96-microwell plate, which were covered with the adhesive strip and then incubated for 2 h at 37 °C.Then, the liquid of each well was poured out, followed by the addition of 100 μl biotin-antibody (1X) in each well for 1 h at 37 °C.After removing the liquid and washing with wash buffer, 100ul horseradish peroxidase (HRP)-avidin (1X) was added to each well and incubated for 1 h at 37 °C.After washing the plate, TMB substrate was added into the microplates and incubated for 15-30 min in a dark environment for color development, and stop solution was used to stop the reaction.The optical density (OD) values were read at 450 and referenced to 570 nm wavelengths within 5 min after adding stop solution by microplate reader (Thermo Fisher Scientific, Boston, USA).
The concentrations of serum IGFBP-1 were obtained by plotting a standard curve with a four-parameter logistic curve manner, and actual serum IGFBP-1 concentration must be multiplied by the dilution factor.Serum samples of patients and normal controls were measured simultaneously in the same batch.All measurements were done in duplicate.

Measurement of tumor markers in clinical use
The concentrations of serum CEA and CA19-9 were measured by an automatic electrochemical luminescence analyzer (Cobas e601, Roche, Germany).All tests of the two tumor markers were performed at the Department of Clinical Laboratory Medicine, the Cancer Hospital of Shantou University Medical College, and operated according to the instrument operating manual.In this study, the recommended clinical cutoff values of CEA, CA19-9 were 5.0 ng/mL and 27 U/mL, respectively.

The level of serum IGFBP-1 in CRC patients and normal controls
Subsequently, we performed ELISA to further evaluate serum levels of IGFBP-1 in CRC patients and normal controls.In total, 328 participants were recruited, 138 patients with CRC and 190 normal controls (Fig. 1).As shown in Supplementary Table S1, the mean age of 138 eligible patients was 58 years (range 26-82 years), of which 78 (56.5%) were males and 60 (43.5%) were females.The control group consisted of 147 (77.37%) males and 43 (22.63%)females aged between 40 and 80 years (mean, 56 years).The mean concentration of serum IGFBP-1 was 1569.455 ± 770.209 ng/mL, 1512.222± 818.971 ng/mL and 719.991 ± 379.340 ng/mL in CRC group (n = 138), early-stage CRC group (n = 68) and normal group (n = 190), respectively (Supplementary Table S2).To get a better view of its distribution and degree of dispersion, the levels of serum IGFBP-1 in three groups were shown in scatter plot (Fig. 2A) and box plot (Fig. 2B).The levels of serum IGFBP-1 in CRC group were higher when compared with normal group, which was confirmed statistically (P < 0.0001).In addition, the difference between early-stage CRC and normal controls is also significant (P < 0.0001).It can be observed that the distribution of CRC and normal controls is different in Fig. 2C.CRC group accounts for more histogram volume on higher concentration while normal group for more lower concentration.In a word, our data demonstrated that serum levels of IGFBP-1 significantly increase in CRC patients.

The diagnostic value of serum IGFBP-1 in CRC and early-stage CRC
To assess the diagnostic value of serum IGFBP-1 for CRC, we performed the ROC analysis to assess the ability of IGFBP-1 to distinguish CRC patients from normal controls.With an optimum diagnostic cutoff of 1258.387ng/ ml, ROC analysis displayed that serum IGFBP-1 achieved an AUC of 0.874 (95% CI 0.835-0.932)for distinguishing CRC from controls CRC (Fig. 3).The specificity and the sensitivity were 90.53% and 63.04%, respectively (Table 1).In addition, with the same cutoff value, IGFBP-1 could discriminate early-stage CRC from normal controls with a slightly lower AUC value of 0.812 (95% CI 0.795-0.908),a specificity of 90.53% and a sensitivity of 58.82% (Fig. 3 and Table 1).For better interpretation on clinical value of serum IGFBP-1, we also analyzed PPV, NPV, PLR and NLR, and the detail results were shown in Table 1.Interestingly, we evaluated the diagnostic performance of IGFBP-1 in CEA-negative, CA 19-9-negative CRC/early CRC.The results showed that IGFBP-1 also showed high diagnostic efficacy in CEA/CA19-9 negative CRC, and similar results were also obtained in CEA/CA19-9 negative early CRC, as shown in Table 2.

Correlation between serum levels of IGFBP-1 and clinical data in CRC
We evaluated the relationship between serum IGFBP-1 and clinicopathological characteristics of CRC patients by comparing serum IGFBP-1 positive rate in all 138 CRC patients.We defined that the positive level of serum IGFBP-1 in CRC patients was higher than 1258.387ng/mL.As shown in Supplementary Tables S3, there were no statistically significant associations between the positive rates of serum IGFBP-1 and depth of tumor invasion,

Comparation of the diagnostic capacity of serum IGFBP-1 with CEA and CA19-9
We found that serum CEA and CA19-9 levels were detected in 126 CRC patients and 45 normal controls, according to the medical records and physical examination data, of which the diagnostic values were used to compare with serum IGFBP-1.The two/three-biomarker panel was established employing a logistical regression model with the predicted probability.As shown in Supplementary Fig. S2A,B and Table 3, the diagnostic efficiency of serum IGFBP-1 was significantly higher than those for CEA, CA19-9 or CEA+CA19-9 for both all-stage CRC and early-stage CRC.More importantly, when compared with IGFBP-1 alone, ROC analysis showed that the combination of IGFBP-1 and CEA, IGFBP-1 and CA19-9 or the three-biomarker panel (IGFBP-1+CEA+CA19-9) had a minute improvement of AUC to distinguish all stage CRC patients from controls (Supplementary Fig. S2A, Table 3).However, compared with detection of IGFBP-1 alone, IGFBP-1+CEA, IGFBP1+CA19-9 or the threebiomarker panel would reduce the diagnostic sensitivity (56.45% vs 40.32%, 48.40% or 43.55%, respectively, Supplementary Fig. S2B, Table 3).

Prognostic value of serum IGFBP-1 in CRC
We investigated whether serum IGFBP-1 could be used to predict prognosis of CRC.The 138 CRC patients recruited in this study were included for prognostic analysis.Using X-tile software, we set 1781.120 ng/mL as the optimal cut-off value to classify high and low expression of IGFBP-1.Univariate Cox analysis showed that age, gender, N stage, M stage, TNM stage and serum IGFBP-1 expression level were associated with prognosis of CRC.Multivariate Cox analysis further demonstrated that M stage (P = 0.010, HR = 3.811, 95%CI 1.373-10.580),TNM stage (P = 0.007, HR = 3.106, 95%CI 1.363-7.077)and serum IGFBP-1 (P = 0.045, HR = 2.043, 95%CI 1.014-4.115)were independent factors to predict the prognosis of CRC (Table 4).According to multivariate Cox proportional hazards regression analysis, the forest map was used to visualize the significant correlation between M stage, TNM stage, IGFBP-1 expression and OS of CRC patients (Supplementary Fig. S3).Furthermore, Kaplan-Meier revealed that the 5-year OS of CRC patients with high serum IGFBP-level was shorter than those with low serum IGFBP-1 level (16% vs 32%), and the difference was statistically significant (P = 0.019) (Fig. 4A).

Nomogram for OS of CRC
In order to better evaluate the prognosis of patients with CRC, the independent prognostic factors of M stage, TNM stage, and serum IGFBP-1 were used to establish a nomogram to forecast 1-, 3-and 5-years OS probability prediction (Fig. 5).From the nomogram, each prognostic factor was assigned a number of risk points, which   www.nature.com/scientificreports/ was obtained by drawing a straight line directly upward to the "points" axis from the corresponding value of the prognostic factor.These points were added together to obtain "total points".The relationship between M stage, TNM stage, serum IGFBP-1 and OS can be visually shown in the nomogram (Fig. 5).The calibration plots were used to evaluate predictive capacity of the nomogram for 1-, 3-, and 5-year OS (Fig. 6).The results showed that the nomogram had a good prediction accuracy for OS.Akaike information criterion (AIC), bayesian information criterion (BIC) and C-index were used to evaluate the goodness-of-fit and discriminative ability of the nomogram.As shown in Table 5, the AIC and BIC of the nomogram were lower than those of TNM stage (291.994vs 305.737; 296.483 vs 307.234, respectively), suggesting that the nomogram had a higher goodness-of-fit for predicting OS.The C-index for the nomogram was 0.714 (95%CI 0.623-0.804),which was higher than that of TNM staging (0.651, 95%CI 0.575-0.727,P = 0.043).Time-dependent C-index analysis also showed that the nomogram showed a good prognostic accuracy for OS when compared with other single prognostic factors (Supplementary Fig. S4).Moreover, the DCA (Fig. 7) showed that the nomogram has higher overall net benefit than the traditional TNM stage systems alone in the majority of the range of threshold probabilities.Taken together, these results demonstrated that the nomogram had a better performance to predict OS of CRC patients when compared to the traditional TNM stage systems.

Risk stratification based on the nomogram
In order to determine whether the CRC patients could be effectively divided into two proposed risk groups based on the nomogram and OS, we calculated each patient's total point, and used the X-tile program to obtain the optimal cut-off value (1781.16ng/ml) to subdivide patients into low-and high-risk subgroups.Kaplan-Meier analysis and log-rank test showed that the high-risk group had shorter OS than those in the group of low-risk (39% vs 66%, P < 0.0001), and the median OS of CRC patients was less than 5 years (Fig. 4B).This stratification demonstrated that the nomogram could effectively divide those patients into the 2 risk subgroups with significant differences in OS.

Discussion
It is well known that endoscopy examination could help identify early-stage CRC 8,34 , but the invasiveness of colonoscopy limits its widely apply as a screening tool in a large number of asymptomatic populations 35 .Besides, the effectiveness of colonoscopy as a screening tool is closely related to the adequate detection and removal of colonic polyps, and the detection rate of colonoscopy is operator dependent in some extent 9 .In addition, some other noninvasive screening tests of CRC including stool blood tests, serum CEA, CA19-9 and blood septin9 gene tests were used to reduce the incidence and mortality of CRC 22,[36][37][38] .Nevertheless, because of the low sensitivity or high cost, none of these methods has been established as a recognized early screening tool 23,[39][40][41] .Therefore, it is urgent to find new methods with high sensitivity and specificity for the early diagnosis of CRC.In recent years, serum protein biomarkers are considered to be the most promising detection methods for population studies and routine clinical work 17,42,43 , because cancer phenotypic features seem most likely to be a direct reflection of changes in protein metabolism and function, which are also the targets of most anticancer drugs in clinical practice 44 , and these serum samples are noninvasive and easily accessible.Furthermore, a growing interest in the clinically potential use of circulating IGFBPs as diagnostic or prognostic biomarkers in cancers 45 .
Our previous studies demonstrated that serum IGFBPs showed good diagnostic or prognostic performance in upper gastrointestinal cancer, including IGFBP-1 31,[46][47][48] .In this study, we found that blood IGFBP-1 mRNA is increased in GEO CRC patient dataset.In addition, in consideration of IGFBP-1 is a secreted protein, we carried out ELISA experiment to detect the levels of serum IGFBP-1 protein, and found that serum IGFBP-1 www.nature.com/scientificreports/expression was significantly increased in CRC, compared to normal controls (P < 0.0001).Meanwhile, serum IGFBP-1 showed a good diagnostic value in all stages of CRC with an AUC of 0.874, a specificity of 90.53% and a sensitivity of 63.04%.In terms of cancer screening, one of the most important attributes of a biomarker should be able to identify early cancers.For early-stage CRC, a certain diagnostic accuracy of serum IGFBP-1 could be observed (AUC 0.812, specificity 90.53% and sensitivity 58.82%).It is reported that CEA or CA-199 did not provide sufficient sensitivity and reliability for the early detection of CRC 21,23,49 .Similarly, our results also suggested that CEA and CA199 showed low diagnostic performance for early-stage CRC.Compared with CEA, CA19-9 or the twobiomarker combined panel (CEA+CA19-9), serum IGFBP-1 has higher sensitivity and AUC value to identify early-stage CRC (Table 3).Therefore, we believe that serum IGFBP-1 protein detection might be helpful for early diagnosis of CRC.Additionally, the combination of IGFBP-1 and CEA, IGFBP-1 and CA19-9, or the threebiomarker combined panel may further increase the diagnostic efficacy, especially with increased sensitivity, but there is no such effect for early-stage patients (Table 3).
Although genetics plays an important role in risk stratification, the risk of developing CRC is also influenced by acquired factors, including age, race, male gender, and dietary habits 40 .Age is one of the risk factors that has been taken into account in most screening recommendations 50 .CRC death rates rose 1.2% per year in people younger than 50 years and 0.6% per year in those aged 50 to 54 years from 2005 to 2019 51 .Whereas a recent study showed similar prevalence at ages 45 to 49 years and 50 to 54 years, which provide clinically useful evidence for optimizing the age in CRC prevention and screening 52 .In our study, we found that the positive rate of serum IGFBP-1 was closely related to patient age.
On one hand, IGFBP-1 is expressed in fetal liver and postpartum tissue, mainly in secretory endometrium, decidua gravidarum and liver.Pathologically, the expression of IGFBP-1 is elevated type 2 diabetes and some cancer patients 25 .Moreover, increasing studies indicate the expression and prognostic value of IGFBP-1 in serum/ tissue of patients with cancer remains equivocal and even controversial [53][54][55][56][57][58] .These results indicated that IGFBP-1 might have different expression pattern and prognostic value in different carcinomas, and the prognostic potential of serum IGFBP-1 in cancers should be further evaluated.So far, there is little literature involving the expression and the diagnostic or prognostic value of serum IGFBP-1 in CRC.Previously, most studies have focused on evaluating the relationship between circulating levels of IGFBP-1 and the risk of CRC, and the results seem to be contradictory.It is reported that higher plasma/serum IGFBP-1 levels were associated with a decreased risk of CRC 59,60 .Analogously, Vidal et al. revealed that lower concentrations of the plasma IGFBP-1 were associated with an increased risk of CRC, whereas higher levels of IGFBP-1 were related with a decreased risk of CRC in men only 61 .On the contrary, Wei et al. found that low blood IGFBP-1 expression was not obviously related with the increased risk of CRC 62 .Palmqvist et al. also reported similar results 63 .On the other hand, many in vitro and in vivo studies support the dual role of IGFBP-1 in tumor proliferation, migration, invasion, and adhesion through both IGF-dependent and IGF-independent molecular mechanisms, suggesting that the effects of IGFBP-1 are cell-specific and dependent on the type of target cells 25,30,56 .Moreover, as described in the literature, IGFBP-1 not only inhibited the invasion and migration of CRC cells SW480 and SW620, but also promoted CRC liver metastasis in mice transplanted with SW480 cells 64 .These findings suggest that IGFBP-1 may have a dual function, playing both positive and negative roles in the progression and metastasis of CRC.Liver metastasis (CLM) occurred only in mice injected with IGFBP-1 overexpressed SW480 cells, but CLM was not found in igfbp1 overexpressed SW620 cells, which may be related to the persistent expression of β-catenin, vimentin, and ZO-1 in IGFBP-1 overexpressed SW480 cells 64 .These results suggest that the role of IGFBP-1 in CRC is complex, but the specific mechanisms have not been clearly elucidated.Because there have been few studies on the role of IGFBP-1 in the development of colorectal cancer and the molecular mechanisms of action.Therefore, in future study, it is important to further explore its biological function in CRC.In our study, we identified that patients with higher serum IGFBP-1 expression had a worse OS.In addition, risk stratification demonstrated that the nomogram could effectively divide those patients into the high-and low-risk subgroups, and patients in the high-risk group had shorter OS.On the other hand, prognostic assessment of CRC patients remains a great clinical challenge.TNM stage system is the primary basis in estimating the prognosis of CRC 65 .However, TNM stage is based fully on the anatomical range of the disease, and may be affected by pathological assessment and tumor heterogeneity, which has limitations for survival analysis of CRC patients 66 .Here, we identified that serum IGFBP-1 was an independent prognostic factor.The nomogram based on serum IGFBP-1, M stage and TNM stage was established, of which the C-index was better than that of the TNM stage alone.A similar result was also observed in time-dependent C-index curve analysis.In addition, the decision curve analysis for 1-, 3-, and 5-year OS showed that the nomogram seemed to have higher overall benefit than TNM stage systems alone.These findings indicated that the nomogram seems to be more accurate in predicting OS than the traditional TNM stage system.In the study, age and lymph node metastasis were excluded after multivariate analysis, possibly due to sample size and clinical characteristics of patients.Therefore, it is necessary to verify whether age and lymph node metastasis are independent prognostic predictors with further large samples in the future.
Several limitations of this study should be also taken into account: in our study, all patients were recruited from individuals with known cancers, and most were diagnosed based on the clinical symptoms of the disease.Nevertheless, the diagnostic sensitivity of IGFBP-1 may be different in individuals with asymptomatic disease or precancerous lesions, which needs to be further verified.Furthermore, these results were assessed in a singleinstitution study, which may lead to bias.Moreover, this study did not analyze the levels of serum IGFBP1 in patients of benign intestinal disease, which should be used as disease controls to assess the efficacy of serum IGFBP-1.Future studies need to address the role of IGFBP-1 in benign intestinal disease.The number of subjects in the control and CRC groups to compare the diagnostic value between IGFBP-1 and the classic clinical tumor markers (CEA and CA19-9) was not balanced, which may reduce statistical power.Whether serum IGFBP-1 had improved performance in the early diagnosis of CRC still needs further validation.Finally, there is a selection bias in the male-to-female ratio within the CRC group and control group.Future studies also need to verify whether the male-to-female ratio difference may influence the results.Therefore, in future study, a study with multicenter and large-scale samples should be completed to further verify the diagnostic and prognostic value of IGFBP-1 in CRC, and need to further evaluate the role of IGFBP1 in benign bowel disease.
In conclusion, our findings offer a convenient and non-invasive for early diagnosis and prediction of outcomes for patients with CRC, as the blood-based IGFBP-1 are easy to obtain from routine admission laboratory tests.Meanwhile, we believe that serum IGFBP-1 detection is not meant to replace endoscopy or traditional prognostic assessment strategy, but contributes to identifying patients who may have CRC at an early stage.Furthermore, our study demonstrated that serum IGFBP-1 is an independent prognostic risk factor for CRC patients, and the construction of nomogram model containing serum IGFBP-1 might improve the accuracy of prognosis prediction.

Figure 3 .
Figure 3. ROC curve analysis in the diagnosis of CRC and early-stage CRC.(A) ROC curve for IGFBP-1 in all stage CRC versus normal control.(B) ROC curve for IGFBP-1 in early stage of CRC versus normal control.(C) Two groups versus normal controls group are in different colors.The area under the red line is 0.5, for reference.

Figure 4 .Figure 5 .
Figure 4. Kaplan-Meier curves for OS of patients with CRC.(A) Survival curve for serum IGFBP-1 with CRC patients, Log-rank test was used to evaluate the significant difference.(B) Survival curve of risk stratification for OS according to prediction of nomogram.The low risk: Total points ≤ 228.75 for OS, the high risk: Total points > 228.75 for OS.Log-rank test was applied to assess the significant difference.The number of people alive at each time point in the high and low IGFBP-1 groups was showed in "number at risk."

Figure 6 .
Figure 6.The calibration plots (A-C) are used to estimate predictive capacity of the nomogram for 1-, 3-, and 5-year OS probability.X-axis was the probability of 1-, 3-or 5-year OS predicted by nomogram.Y-axis was the actual OS of the patients included in the study.

Figure 7 .
Figure 7. Decision curve analysis the predictive accuracy of M stage, TNM stage, IGFBP-1 and nomogram in CRC patients.The decision curve of 1-(A), 3-(B), and 5-(C) year OS.The y-axis represents the net benefit, which is calculated by summing the benefits (true positive results) and subtracting the harms (false positive results).The horizontal line represents the assumption that no deaths happen.

Table 1 .
Evaluation of the detection value of IGFBP-1 in the diagnosis of CRC.

Table 2 .
Evaluation of the detection value of IGFBP-1 in the diagnosis of CEA/CA19-9-negative early CRC and CRC.

Table 3 .
Diagnostic performance of different biomarkers in 126 CRC patients and 45 normal controls.

Table 4 .
Cox analysis of OS for CRC.

Table 5 .
The C-index of IGFBP-1, M stage, TNM stage and nomogram for prediction of OS in CRC.AIC Akaike information criterion, BIC Bayesian information criterion.